Biosensors

Abstract

The invention relates to biosensors, more particularly to protein biosensors and their use to indicate the formation of denaturing ice crystals in samples. The invention includes the use of a protein as a biosensor to indicate denaturing ice-crystal formation in a sample, wherein the protein is denaturable by denaturing ice crystals from a non-denatured state to a denatured state and wherein said non-denatured and denatured states are associated with detectably different indications, e.g. colour, fluorescence, bioluminescence, protein activity, etc. Biosensor compositions according to the invention preferably comprise a protein comprising a ligand-binding domain and a ligand bindable by the ligand-binding domain. Particularly good results are obtained using proteins, especially fluorescent proteins, having at least one polyhistidine tag and a ligand comprising imidazole or an equivalent polyhistidine-binding component. For example, polyhistidine-tagged Ds Red Express2 (Ds Red2) is especially preferred for use in the invention. On denaturation by denaturing ice crystals, this protein undergoes a marked change of colour indication from red to colourless. The invention is suitable for indicating the formation of denaturing ice crystals in a range of different samples susceptible to ice crystal damage, e.g. foodstuffs, pharmaceutical preparations, laboratory reagents, cells, tissues, etc.

1 . The use of a protein as a biosensor to indicate denaturing ice-crystal formation in a sample, wherein the protein is denaturable by denaturing ice crystals from a non-denatured state to a denatured state and wherein said non-denatured and denatured states are associated with detectably different indications, said protein comprising a ligand-binding domain. 
     
     
         2 . The use according to  claim 1 , wherein the protein is at least partially irreversibly denaturable by denaturing ice crystals. 
     
     
         3 . (canceled) 
     
     
         4 . (canceled) 
     
     
         5 . The use according to  claim 1 , wherein the non-denatured and denatured states of the protein are associated with detectably different colour indications. 
     
     
         6 . The use according to a  claim 1 , wherein the protein is a fluorescent protein. 
     
     
         7 . The use according to  claim 6 , wherein the protein comprises a protein selected from: DsRed, DsRed2, EGFP, GFP, ECFP, Cerulean, CyPet, YFP, Citrine, Venus, YPet, TagRFP1, mKate2, TurboRFP, TurboFP635 and mCherry, and analogues and derivatives thereof. 
     
     
         8 . The use according to  claim 7 , wherein the protein comprises DsRed2. 
     
     
         9 . (canceled) 
     
     
         10 . The use according to  claim 1 , wherein the protein is a fusion protein. 
     
     
         11 . (canceled) 
     
     
         12 . (canceled) 
     
     
         13 . The use according to  claim 1 , wherein the ligand-binding domain and its corresponding ligand are selected from: polyhistidine and imidazole, glutathione S-transferase (GST) and gluthathione, streptavidin and biotin, luciferase and luciferin, and analogues and derivatives thereof. 
     
     
         14 . The use according to  claim 13 , wherein the ligand and ligand-binding domain comprise imidazole, or an analogue, derivative or functional equivalent thereof, and polyhistidine, respectively. 
     
     
         15 . The use according to  claim 14 , wherein the concentration of ligand is 10-500 mM, 25-400 mM, 50-300 mM, 75-250 mM, 100-200 mM, 125-175 mM or around 150 mM; and the concentration of the polyhistidine-tagged protein is 0.1-5 mg/ml, 0.25-4 mg/ml, 0.5-3 mg/ml, 0.75-2 mg/ml, 1-1.5 mg/ml or around 1 mg/ml. 
     
     
         16 . The use according to  claim 1 , wherein the protein is provided with at least one metal binding domain. 
     
     
         17 . The use according to  claim 16 , wherein the metal binding domain comprises the peptide sequence YIDTNNDGWYEGDELLA (SEQ ID NO: 1). 
     
     
         18 . The use according to  claim 1 , wherein the sample is selected from one or more of the following: pharmaceuticals, antibodies, enzymes, biopharmaceuticals, laboratory reagents, tissues, cells, cell research products, blood, blood products and food products. 
     
     
         19 . A biosensor composition for indicating denaturing ice-crystal formation in a sample, the composition comprising a protein, wherein the protein is denaturable by denaturing ice crystals from a non-denatured state to a denatured state and wherein said non-denatured and denatured states are associated with detectably different indications, said protein comprising a ligand-binding domain. 
     
     
         20 . (canceled) 
     
     
         21 . (canceled) 
     
     
         22 . A biosensor composition according to  claim 19 , wherein the biosensor composition further comprises
 a ligand bindable by the ligand-binding domain.   
     
     
         23 . A biosensor composition according to  claim 19 , wherein the biosensor composition further comprises water. 
     
     
         24 . A biosensor composition according to  claim 23 , wherein the biosensor composition further comprises one or more of the following components: PBS, NTA, NTA derivatives, 3-{[tris(hydroxylmethyl)methyl]amino}propanesulfonic acid, N,N-bis-(2-hydroxyethyl)glycine, tris(hydroxymethyl)methylamine, N-tris-(hydroxyl-methyl)methylglycine, 4-2-hydroxyethyl-1-piperazineethanesulfonic acid, 3-(N-morpholino)propanesulfonic acid, piperazine-N,N′-bis(2-ethanesulfonic acid), saline sodium citrate, 2-(N-morpholino)ethanesulfonic acid, acetic acid, sodium acetate, K 2 HPO 4 , KH 2 PO 4 , KCl and EDTA. 
     
     
         25 . A biosensor composition according to  claim 19 , wherein the protein is at least partially irreversibly denaturable by denaturing ice crystals. 
     
     
         26 . (canceled) 
     
     
         27 . (canceled) 
     
     
         28 . A biosensor composition according to  claim 19 , wherein the non-denatured and denatured states of the protein are associated with detectably different colour indications. 
     
     
         29 . A biosensor composition according to  claim 19 , wherein the protein is a fluorescent protein. 
     
     
         30 . A biosensor composition according to  claim 29 , wherein the protein comprises a protein selected from: DsRed, DsRed2, EGFP, GFP, ECFP, Cerulean, CyPet, YFP, Citrine, Venus, YPet, TagRFP1, mKate2, TurboRFP, TurboFP635 and mCherry, and analogues and derivatives thereof. 
     
     
         31 . A biosensor composition according to  claim 30 , wherein the protein comprises DsRed2. 
     
     
         32 . (canceled) 
     
     
         33 . A biosensor composition according to  claim 19 , wherein the protein is a fusion protein. 
     
     
         34 . A biosensor composition according to  claim 22 , wherein the ligand and ligand-binding domain are selected from: imidazole and polyhistidine, glutathione and glutathione S-transferase (GST), biotin and streptavidin, luciferin and luciferase, and analogues and derivatives thereof. 
     
     
         35 . A biosensor composition according to  claim 34 , wherein the ligand and ligand-binding domain comprise imidazole, or an analogue, derivative or functional equivalent thereof, and polyhistidine, respectively. 
     
     
         36 . A biosensor composition according to  claim 35 , wherein the concentration of ligand is 10-500 mM, 25-400 mM, 50-300 mM, 75-250 mM, 100-200 mM, 125-175 mM or around 150 mM; and the concentration of the polyhistidine-tagged protein is 0.1-5 mg/ml, 0.25-4 mg/ml, 0.5-3 mg/ml, 0.75-2 mg/ml, 1-1.5 mg/ml or around 1 mg/ml. 
     
     
         37 . A biosensor composition according to  claim 19 , wherein the protein is provided with at least one metal binding domain. 
     
     
         38 . A biosensor composition according to  claim 37 , wherein the metal binding domain comprises the peptide sequence YIDTNNDGWYEGDELLA (SEQ ID NO: 1). 
     
     
         39 . A biosensor composition according to  claim 19 , wherein the sample is selected from one or more of the following: pharmaceuticals, antibodies, enzymes, biopharmaceuticals, laboratory reagents, tissues, cells, cell research products, blood, blood products and food products. 
     
     
         40 . A method of indicating denaturing ice-crystal formation in a sample stored under storage conditions, the method comprising:
 (i) storing under said storage conditions a biosensor composition comprising a protein, wherein the protein is denaturable by denaturing ice crystals from a non-denatured state to a denatured state and wherein said non-denatured and denatured states are associated with detectably different indications and wherein said protein comprises a ligand-binding domain; and water; and   (ii) detecting any change of indication.   
     
     
         41 . A method according to  claim 40 , wherein the protein is at least partially irreversibly denaturable by denaturing ice crystals. 
     
     
         42 . (canceled) 
     
     
         43 . (canceled) 
     
     
         44 . A method according to  claim 40 , wherein the non-denatured and denatured states of the protein are associated with detectably different colour indications. 
     
     
         45 . A method according to  claim 40 , wherein the protein is a fluorescent protein. 
     
     
         46 . A method according to  claim 45 , wherein the protein comprises a protein selected from: DsRed, DsRed2, EGFP, GFP, ECFP, Cerulean, CyPet, YFP, Citrine, Venus, YPet, TagRFP1, mKate2, TurboRFP, TurboFP635 and mCherry, and analogues and derivatives thereof. 
     
     
         47 . A method according to  claim 46 , wherein the protein comprises DsRed2. 
     
     
         48 . (canceled) 
     
     
         49 . A method according to  claim 40 , wherein the protein is a fusion protein. 
     
     
         50 . (canceled) 
     
     
         51 . (canceled) 
     
     
         52 . A method according to  claim 40 , wherein the accessory component is a ligand and the protein is provided with at east one corresponding ligand binding domain selected from: polyhistidine and imidazole, glutathione S-transferase (GST) and gluthathione, streptavidin and biotin, luciferase and luciferin, and analogues and derivatives thereof. 
     
     
         53 . A method according to  claim 52 , wherein the ligand and ligand-binding domain comprise imidazole, or an analogue, derivative or functional equivalent thereof, and polyhistidine, respectively. 
     
     
         54 . A method according to  claim 53 , wherein the concentration of ligand is 10-500 mM, 25-400 mM, 50-300 mM, 75-250 mM, 100-200 mM, 125-175 mM or around 150 mM; and the concentration of the polyhistidine-tagged protein is 0.1-5 mg/ml, 0.25-4 mg/ml, 0.5-3 mg/ml, 0.75-2 mg/ml, 1-1.5 mg/ml or around 1 mg/ml. 
     
     
         55 . A method according to  claim 40 , wherein the protein is provided with at least one metal binding domain. 
     
     
         56 . A method according to  claim 55 , wherein the metal binding domain comprises the peptide sequence YIDTNNDGWYEGDELLA (SEQ ID NO: 1). 
     
     
         57 . A method according to  claim 40 , wherein the sample is selected from one or more of the following: pharmaceuticals, antibodies, enzymes, biopharmaceuticals, laboratory reagents, tissues, cells, cell research products, blood, blood products and food products. 
     
     
         58 . A method according to  claim 40 , wherein the biosensor composition further comprises one or more of the following components: PBS, NTA, NTA derivatives, 3-{[tris(hydroxylmethyl)methyl]amino}propanesulfonic acid, N,N-bis-(2-hydroxyethyl)glycine, tris(hydroxymethyl)methylamine, N-tris-(hydroxyl-methyl)methylglycine, 4-2-hydroxyethyl-1-piperazineethanesulfonic acid, 3-(N-morpholino)propanesulfonic acid, piperazine-N,N′-bis(2-ethanesulfonic acid), saline sodium citrate, 2-(N-morpholino)ethanesulfonic acid, acetic acid, sodium acetate, K 2 HPO 4 , KH 2 PO 4 , KCl and EDTA. 
     
     
         59 . A method according to  claim 40 , wherein the method further comprises a step of exposing the protein to UV light. 
     
     
         60 . A method according to  claim 40 , wherein the biosensor composition is introduced into or otherwise combined with the sample prior to storage. 
     
     
         61 . A method according to  claim 40 , wherein the biosensor composition is stored separately from but in conjunction with the sample. 
     
     
         62 . A method according to  claim 61 , wherein the biosensor composition is stored separately in or on containment means containing the sample. 
     
     
         63 . A method according to  claim 62 , wherein the biosensor composition is applied to packaging of the sample. 
     
     
         64 . (canceled) 
     
     
         65 . A method according to  claim 62 , wherein the biosensor composition forms part of a bar code or other identification means. 
     
     
         66 . (canceled) 
     
     
         67 . A method according to  claim 40 , wherein the biosensor composition further comprises a ligand bindable by the ligand-binding domain.